Mammalian myotrophin/V-1 is a 13 kDa, ankyrin-repeat protein that binds Capping Protein (CP) in vitro with an affinity of 20 nM, creating a 1:1 complex (CP: V-1) that has no affinity for the barbed end. V-1 has the potential, therefore, to influence actin polymerization in vivo by reducing the extent of barbed end capping. Dictyostelium discoideum (D.d.) contains a single gene encoding a 13.2 kDa protein with high sequence similarity to mouse V-1. Like mouse V-1, Dd V-1 binds CP tightly, is present in cells at a three-fold molar excess over CP, and no longer binds CP when a function-blocking point mutation (FBM) is introduced into the first ankyrin loop (FBM Dd V-1). Consistent with V-1s ability to sequester CP, over-expression of Dd V-1 results in an elevation in total cellular F-actin content that scales positively with the degree of over-expression. Importantly, over-expression of FBM Dd V-1 does not alter cellular F-actin levels, arguing that the effect of V-1 over-expression is due to its ability to sequester CP. The over-expression of Dd V-1, but not FBM-Dd V-1, also induces the formation of actin-rich, filopodial-like structures that scales positively with the degree of over-expression. In contrast to over-expression, Dd V-1 null cells created by homologous recombination exhibit a large decrease in cellular F-actin content. Moreover, these cells exhibit significant decreases in growth rate, macropinocytosis rate, chemotactic streaming efficiency, polarity during migration, and random motility rate. Importantly, these defects are rescued by wild type V-1 but not by FBM Dd V-1. Together, these results argue that V-1 plays a major role in regulating actin assembly in cells. Moreover, the fact that FBM V-1 neither induces over expression phenotypes nor rescues null cell phenotypes argues that V-1 exerts its effects on the actin cytoskeleton by buffering cellular CP. We conclude, therefore, that reductions and elevations in cellular CP activity caused by the over-expression and knock out of V-1, respectively, result in changes in cellular actin content that are consistent with CPs barbed end capping activity. Moreover, the functional consequences of these changes, which involve alterations in numerous cellular activities that dependent on actin assembly, indicate that V-1 is physiologically important.